1	Strategies for Producing and Maintaining Clean Cultures Barbara M. Reed USDA-ARS National Clonal Germplasm Repository
2	Overview Strategies for producing clean cultures Selecting plant materials Laboratory procedures Indexing explants Treating internal contaminants Strategies for maintaining clean cultures Detecting contaminants Laboratory environment Laboratory protocols
3	Strategies for producing clean cultures Growth condition and health of the mother plant Choice of plant tissue used to initiate cultures Laboratory procedures Use of specialized detection media Check plant cultures at key steps to limit spread of contaminants
4	Growth condition and health of the mother plant Healthy plant – virus tested– one of the most important steps in getting a clean culture Not healthy – don’t bother until you revitalize it -only vigorous plants will make vigorous cultures
5	Growth condition and health of the mother plant Indoor plant – best chances for success Outdoor plant – Manipulate for a good outcome
6	Growth condition and health of the mother plant Herbaceous – use only actively growing tips Woody – force new growth in a clean environment Dormant Evergreen Tropical
7	Choice of plant tissue used to initiate cultures Aerial – cleaner if further from the soil Terrestrial – often heavily Infested Use similar tactics to woody plants, force new clean growth Terminal – meristems may be used to avoid contaminants Basal – more likely to be contaminated
8	Choice of plant tissue used to initiate cultures Young – vigorous and fast growing Old – can’t outgrow the contaminant
9	Laboratory procedures Surface sterilization procedures Bleach Alcohol – can harbor bacteria Other sterilants Surfactants
10	Laboratory procedures Sterile technique Unobstructed air flow from filter to plants Instrument sterilization timing and type Container handling Cutting surface Operator training
11	Use of specialized media to detect contaminants Minimal medium ½ MS at pH 6.8 Nutrient broth AC broth Enriched medium 523 medium (Viss) Peptone and yeast extract Leifert and Waites
12	Use of specialized media to detect contaminants Liquid Submerged Partially submerged Rafts or glass beads Semisolid Standard media Many selective media
13	Check plant cultures at key steps during initiation Initial screening of explants May require several steps We use ½ X liquid MS at pH 6.9 for 1 week Move to solid medium and streak on Viss (523) Watch for 3 weeks
14	Check plant cultures at key steps during initiation Variation of indexing medium Alternate media to pick up diverse organisms Use specialized media if specific organisms are common i.e. Pseudomonas medium, King’s B, Agrobacterium specific, Peptone sucrose broth (Xanthomonads)
15	Treating internal contaminants Internal contaminants can be deep within the plant and often will only appear well into the multiplication phase Try screening explants for clean cultures first and use cleanup as a last resort
16	Treating internal contaminants Antibiotic treatments Work best on newly collected explants Usually are not successful on obviously contaminated cultures
17	Strategies for maintaining clean cultures Check cultures at key steps to limit spread of contaminants Clean growth and transfer rooms Proper sterile technique Careful analysis of all procedures Elimination of sources of mites and insects
18	Check plant cultures at key steps to limit spread of contaminants Timing of detection steps at or before Initiation Multiplication Rooting Vary detection medium Use specialized media if specific organisms are common Identify the organism if one is predominant
19	Clean growth and transfer rooms Wet mop to limit dust Use easily cleaned shelving Limit entry from exterior doors Have dedicated mops for the lab Autoclave contaminated containers before washing Evaluate rooms for mold sources and remedy them
20	Proper sterile technique Cutting surface Operator training Hands Clothing and hair The “no reach” zone Container handling Unobstructed air flow from filter to plants Instrument sterilization timing and type
21	Careful analysis of all procedures Evaluate all procedures for their contribution to contamination in the cultures Study air flow and building heating and cooling systems for contaminant sources Institute schedules for changing and checking room and transfer-hood filters
22	Careful analysis of all procedures Monitor autoclave function – pressure and temperature Use autoclave tape to prevent mixups Protect unused media from airborne contamination
23	Elimination of mite and insect sources Limit windows and doors in lab area Limit employee exposure to outside plants Provide separate area for storing coats, field clothing, shoes Wet mop area on a regular schedule Treat source plants with pesticides if they might harbor thrips or mites Use sticky traps on growth room shelves
24	Summary Concentrate on healthy stock plants Use screening medium to select clean explants Place a high priority on sterile technique Get an impartial evaluation of your laboratory to identify problem areas Evaluate all laboratory procedures Eliminate sources of contamination